Tyrosine kinases are a class of enzymes that catalyze the transfer of the terminal phosphate of adenosine triphosphate (ATP) to tyrosine residues in protein substrates. Tyrosine kinases are believed, by way of substrate phosphorylation, to play critical roles in signal transduction for a number of cell functions. In fact, tyrosine kinases have been shown to be important contributing factors in cell proliferation, carcinogenesis, and cell differentiation. Tyrosine kinases can be categorized as either receptor tyrosine kinases or non-receptor tyrosine kinases.
Receptor tyrosine kinases are key regulators of intercellular communication that controls cell growth, proliferation, differentiation, survival, and metabolism. About 20 different receptor tyrosine kinase families have been identified that share a similar structure, namely an extracellular binding site for ligands, a transmembrane region, and an intracellular tyrosine kinase domain (see, e.g., Ullrich et al., Cell, 61:203-212 (1990); Pawson, Eur. J. Cancer, 38(Supp 5):S3-S10 (2002)). For example, the EGFR family of receptor tyrosine kinases comprises EGFR/HER1/ErbB1, HER2/Neu/ErbB2, HER3/ErbB3, and HER4/ErbB4. Ligands of this family of receptors include epithelial growth factor (EGF), TGF-α amphiregulin, HB-EGF, betacellulin, and heregulin. Other receptor tyrosine kinase families include the PDGF family, the FLK family, and the insulin family of receptors.
Extracellular ligand binding of receptor tyrosine kinases induces or stabilizes receptor dimerization, leading to increased kinase activity. The intracellular catalytic domain displays the highest level of conservation among receptor tyrosine kinases, includes the ATP-binding site that catalyzes receptor autophosphorylation of cytoplasmic tyrosine residues, and serves as the docking site for Src homology 2 (SH2)- and phosphotyrosine-binding (PTB) domain-containing proteins such as Grb2, Shc, Src, Cb 1, and phospholipase C-γ. These proteins subsequently recruit additional effectors containing SH2-, SH3-, PTB-, and pleckstrin-homology (PH) domains to the activated receptor, which results in the assembly of signaling complexes at the membrane and the activation of a cascade of intracellular biochemical signals. The most important downstream signaling cascades activated by receptor tyrosine kinases include the Ras/Raf/mitogen activated protein (MAP) kinase pathway, the phosphoinositide 3-kinase/Akt pathway, and the JAK/STAT pathway. The complex signaling network triggered by receptor tyrosine kinases eventually leads either to activation or repression of various subsets of genes and thus defines the biological response to a given signal.
The activity of receptor tyrosine kinases and their signaling cascades is precisely coordinated and tightly controlled in normal cells. However, deregulation of the receptor tyrosine kinase signaling system, either by stimulation through growth factor and/or through genetic alteration, produces deregulated tyrosine kinase activity. These aberrations generally result in receptor tyrosine kinases with constitutive or strongly enhanced kinase activity and subsequent signaling capacity, which leads to malignant transformation. Therefore, they are frequently linked to human cancer and also to other hyperproliferative diseases such as psoriasis (Robertson et al., Trends Genet., 16:265-271 (2000)). The most important mechanisms leading to constitutive receptor tyrosine kinase signaling include overexpression and/or gene amplification, genetic alterations such as deletions and mutations within the extracellular domain or catalytic site, and autocrine-paracrine stimulation through aberrant growth factor loops.
More particularly, gene amplification and/or overexpression of receptor tyrosine kinases occurs in many human cancers, which might increase the response of cancer cells to normal growth factor levels. Additionally, overexpression of a specific receptor tyrosine kinase on the cell surface increases the incidence of receptor dimerization, even in the absence of an activating ligand. In many cases, this results in constitutive activation of the receptor tyrosine kinase, leading to aberrant and uncontrolled cell proliferation and tumor formation. For example, EGFR/HER1/ErbB1 is frequently overexpressed in non-small cell lung, bladder, cervical, ovarian, kidney, and pancreatic cancer as well as in squamous cell carcinomas of the head and neck (Hong et al., Oncol. Biother., 1:1-29 (2000)). The predominant mechanism leading to EGFR overexpression is gene amplification, with up to about 60 copies per cell reported in certain tumors (Libermann et al., Nature, 313:144-147 (1985)). In general, elevated levels of EGFR expression are associated with high metastatic rate and increased tumor proliferation (Pavelic et al., Anticancer Res., 13:1133-1138 (1993)). Therefore, receptor tyrosine kinases such as EGFR are recognized as attractive targets for the design and development of compounds that can specifically inhibit their tyrosine kinase activity in cancer cells.
Small molecule tyrosine kinase inhibitors compete with the ATP-binding site of the catalytic domain of target tyrosine kinases. Such inhibitors are generally orally active and have a favorable safety profile that can easily be combined with other forms of cancer therapy. Several tyrosine kinase inhibitors have been identified to possess effective antitumor activity and have been approved or are in clinical trials. These include gefitinib (Iressa®), sunitinib (Sutent®; SU11248), erlotinib (Tarceva®; OSI-1774), lapatinib (GW-572016), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib mesylate (Gleevec®; STI571), and leflunomide (SU101). Although tyrosine kinase inhibitors represent a new class of targeted therapy that interferes with specific cell signaling pathways and allows target-specific therapy for selected malignancies, there is currently a lack of tumor response to these inhibitors in the general population. For example, only about 10% of patients with non-small cell lung cancer in whom standard therapy failed respond to the EGFR inhibitor gefitinib (Fukuoka et al., J. Clin. Oncol., 21:2237-2246 (2003); Kris et al., JAMA, 290:2149-2158 (2003)). In addition, patients may be at risk of toxicity to tyrosine kinase inhibitors. Furthermore, tyrosine kinase inhibitor therapy is typically very expensive in comparison to conventional chemotherapy. Moreover, resistance to tyrosine kinase inhibitors can manifest during treatment, and sometimes a particular inhibitor becomes wholly ineffective in certain patients.
As a result, due to the high cost of tyrosine kinase inhibitor therapy, the small percentage of responders, the risk of toxic side-effects, and the possibility of developing resistance during treatment, it is imperative to prescribe tyrosine kinase inhibitors only to those patients for whom such therapy will have some benefit. Thus, there is a need in the art for methods that utilize a combination of biomarkers to predict a patient's response to tyrosine kinase inhibitors such as EGFR inhibitors. There is also a need in the art for methods that utilize a combination of biomarkers to identify patients who are at greater risk of developing toxicity to tyrosine kinase inhibitors and to reduce the toxic effects of tyrosine kinase inhibitors in patients already receiving the drug. There is a further need in the art for methods that utilize a combination of biomarkers to identify patients with acquired resistance to tyrosine kinase inhibitor therapy in recurring tumors. The present invention satisfies these needs and provides related advantages as well.